P998: REGULATION OF BCL-X SPLICING BY SRC KINASES INHIBITORS IN MYELOPROLIFERATIVE NEOPLASMS
Authors: J. Petiti, F. Itri, C. Fava, D. Cilloni
Journal: HemaSphere (2022)

Background: Myeloproliferative neoplasms (MPN) are a group of incurable diseases that include essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Both ET and PV are associated with prolonged clinical courses, with symptoms that affect their quality of life, and risk of progression to MF and acute leukemia. MF is the MPN subtype with the most adverse prognosis and is associated with a high risk of leukemic transformation. The FDA approved the use of ruxolitinib, a JAK1/2 kinase inhibitor, that proved to be effective in reducing symptoms and spleen volume, but not in inducing complete molecular remission. Although hematopoietic stem cell transplantation involves a high risk of mortality, it is nowadays the only curative treatment for MF. Therefore, it is relevant to identify new therapeutic strategies to improve the clinical outcome of MPN. Recently, there has been a growing interest in Bcl‐xL, the long isoform encoded by alternative splicing of the Bcl‐x gene, that acts as an anti‐apoptotic regulator. Bcl-xL appears to be up-regulated in MPN patients and we previously showed that its modulation correlates with the clinical severity of MPN subgroups. We also demonstrated that ABT-737, a specific Bcl-xL inhibitor, has a synergistic effect in combination with Ruxolitinib in HEL cell lines and MPN patients’ cells, suggesting that Bcl-xL inhibition could increase the power of treatment (Petiti et al. J Cell Mol Med. 2020). Unfortunately, ABT-737 is characterized by poor bioavailability, so a new approach to target Bcl-xL must be investigated. Paronetto et al. found that tyrosine phosphorylation of Sam68 in cancer cells may protect them from apoptosis by altering the Bcl-xS/L ratio in favor of Bcl-xL. Intriguingly, c-Src is one of the tyrosine kinases that phosphorylate Sam68 and several papers indicated its involvement in the MPN pathogenesis, without explaining the molecular mechanism. Aims: We aimed to molecular investigate the mechanism linking c-Src kinase activity with the deregulation of Bcl-x splicing in MPN, intending to consider c-Src kinase inhibitors, widely bioavailable, as indirect modulators of Bcl-xL to improve MPNs outcome. Methods: HEL cell lines, JAK2 V617F-mutated in homozygosis, has been incubated with different concentrations of ruxolitinib (JAK1/2 inhibitor) and dasatinib (SRC kinases inhibitor) at different concentrations and time points. Then, proliferation, and apoptosis were evaluated. Subsequently, the c-Src pathway, Sam68, and Bcl-xL/S ratio were investigated at both RNA and protein levels. Results: Performing in vitro study on HEL cell lines, we identified that dasatinib induced apoptosis and inhibition of proliferation in HEL cell lines, with a synergistic effect in combination with ruxolitinib. We also showed that dasatinib alone inhibited c-Src and Sam68 phosphorylation and down-modulated Bclx-L protein expression. Combination treatment with ruxolitinib and dasatinib increased both pro-apoptotic Cleaved Caspase 3 and BAX protein expression. Furthermore, we noted that treatment with ruxolitinib alone decreases the mRNA levels of Bclx-L, but also of the Bcl-xS isoform. On the contrary, combined drug therapy modulated the Bcl-xL/S mRNA ratio in favor of the pro-apoptotic Bcl-xS isoform. Summary/Conclusion: Although further studies will be necessary to better understand the c-Src role in Bcl-x splicing regulation in MPN, our preliminary data suggest that indirect downmodulation of Bcl-xL through SRC kinases inhibitors in combination with standard therapy could be useful in the future for the treatment of MPN patients.